Characterization of the inflammatory phenotype of Mycobacterium avium subspecies paratuberculosis using a novel cell culture passage model

Abstract

Understanding the pathogenic mechanisms of Mycobacterium avium subspecies paratuberculosis (MAP) and the host responses to Johne's disease is complicated by the multi-faceted disease progression, late-onset host reaction and the lack of available ex vivo infection models. We describe a novel cell culture passage model that mimics the course of infection in vivo. The developed model simulates the interaction of MAP with the intestinal epithelial cells, followed by infection of macrophages and return to the intestinal epithelium. MAP internalization triggers a minimal inflammatory response. After passage through a macrophage phase, bacterial reinfection of MDBK epithelial cells, representing the late phase of intestinal mucosal infection, is associated with increased synthesis of the pro-inflammatory transcripts of IL-6, CCL5, IL-8 and IL-18, paired with decreased levels of TGFb. Transcriptome analysis of MAP from each stage of epithelial cell infection identified increased expression of lipid biosynthesis and lipopeptide modification genes in the inflammatory phenotype of MAP. Total lipid analysis by HPLC-ES/MS indicates different lipidomic profiles between the two phenotypes and a unique set of lipids composing the inflammatory MAP phenotype. The presence of selected upregulated lipid-modification gene transcripts in samples of ileal tissue from cows diagnosed with Johne's disease supports and validates the model. By using the relatively simple cell culture passage model, we show that MAP alters its lipid composition during intracellular infection and acquires a pro-inflammatory phenotype, which likely is associated with the inflammatory phase of Johne's disease.

Mycobacterium avium subsp. paratuberculosis (Map) Fatty Acids Profile Is Strain-Dependent and Changes Upon Host Macrophages Infection

“…As Mtb, Map adapts to the intracellular environment in the macrophage, where the bacteria overcome exposure to cationic antimicrobial peptides, reactive oxygen species (ROS) and nutrient starvation, via the regulation of genes affecting its envelope's composition (Thirunavukkarasu et al, 2014). In fact, a different lipid profile was previously observed in the envelope of intracellular Map after 1 h of infection (Alonso-Hearn et al, 2010; Everman et al, 2015). The full significance of these alterations in lipid metabolism had to be yet revealed to identify the issues facing Map within host macrophages.…”

Pathogenesis, Molecular Genetics, and Genomics of Mycobacterium avium subsp. paratuberculosis, the Etiologic Agent of Johne’s Disease

“…Once inside phagocytic cells, the ability of MAP to survive and replicate within these phagocytic cells plays a key role in pathogenesis ( 39 , 40 ). Moreover, use of a culture passage model showed that MAP lipid composition changes in macrophages developing a pro-inflammatory phenotype ( 41 ).…”

Antibodies against invasive phenotype-specific antigens increase Mycobacterium avium subspecies paratuberculosis translocation across a polarized epithelial cell model and enhance killing by bovine macrophages

“…Unfortunately, the results obtained from the pipeline of determining host toxicity and vaccine efficacy from in vitro cultures and mouse models did not translate in a successful vaccine trial in ruminant hosts due to unappreciated differences in immunity and pathogenesis of the infection between animal species (Hines et al, 2014 ). Furthermore, the phenotypic changes that occur within MAP during infection (Everman et al, 2015 ) or during exposure to different environmental or host reservoirs (Cirillo et al, 1997 ; Patel et al, 2006 ; Alonso-Hearn et al, 2010 ) may result in ineffective vaccine efficacy. It is possible that due to the incorrect focus of vaccine development, chosen vaccine candidates are not representative of the most relevant antigens during the stages of Johne's disease in the animal.…”